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I. Background
In non-apoptotic, healthy cells, cellular energy produced during mitochondrial respiration, is maintained as an electrochemical gradient that constitutes a high mitochondrial transmembrane potential (ΔΨmhigh). This membrane potential enables the cell to drive the synthesis of ATP, and its disruption is associated with uncoupling of oxidative phosphorylation, generation of superoxide free radicals and release of mitochondrial matrix-associated Ca2+ into the cytosol. Additionally, leakage of key apoptotic mitochondrial proteins such as cytochrome C, HtrA2/Omi, SMAC/Diablo and apoptosis inducing factor (AIF) have been associated with loss of ΔΨm. Thus, decreases in membrane potential have been used as a characteristic apoptotic marker. Trevigen’s MitoShiftTM Kit uses a unique cationic dye (tetramethylrhodamine ethylester) that can be used to visualize mitochondria in cells. Although the maximum absorption/emission of this dye is 549 nm and 575 nm respectively, an excitation of 488 nm and emission at 565 nm was used successfully to monitor mitochondrial function in intact cells. In healthy cells, the mitochondria appear as punctuate, perinuclear, yellow-red staining as observed by fluorescence confocal microscopy or by flow cytometry. Upon disruption of the mitochondrial potential, the dye shifts out of the mitochondria into the cytoplasm, where it produces diffuse orange fluorescence. Mitochondrial events can be monitored at the single mitochondrial level, and the distribution of the dye across the mitochondrial membrane can be analyzed to quantitate DYm using a ratiometric approach based on the Nernst equation. Trevigen’s MitoShiftTM Kit has been used to evaluate the influence of drugs or cytotoxins on cellular mitochondria or to quickly estimate their effects on a cell population or detect apoptosis in known models.

II. Precautions and Limitations
1. For Research Use Only. Not for use in diagnostic procedures.
2. The physical, chemical, and toxicological properties of the products contained within the MitoShift Kit may not yet have been fully investigated. Therefore, Trevigen recommends the use of gloves, lab coats, and eye protection while using any of these chemical reagents. Trevigen assumes no liability for damage resulting from handling or contact with these products. MSDS sheets are available.

V. Assay Protocol
NOTE: Mitochondrial membrane potential is pH sensitive. For optimal results, ensure that all reagents used are buffered between pH 7.0 and 8.0 and analyze rapidly. Cell culture media may be used in place of the reaction buffer provided.
A. Microscopic/Confocal Observation
a. Staining of Monolayer Cells
1. Dilute the 10X Reaction Buffer to 1X with distilled water and prewarm to room temperature before use. To increase the stability of the dye for viewing under the microscope, add 20 ml of Stabilizer Solution for every 1 ml of 1X
Reaction Buffer prepared.
2. To dilute the MitoShift™ dye, add 1 ml of dye to 10 ml of 1X Reaction Buffer prepared in the previous step (with or without the Stabilizer solution). Vortex to homogenize. Again, add 1 ml from this solution to 1000 ml of 1X reaction buffer and vortex again. A starting concentration of 100 nM (1:10,000 dilution) is suggested, although optimization may be required depending upon the cell type under investigation.
3. Remove media and wash cells in 1X reaction buffer.
4. Cover the cells with diluted MitoShift™ solution prepared in step 2.

5. Incubate at room temperature, in the dark for 30-90 minutes to let the dye equilibrate. A humidity chamber (please see Appendix) may be used to avoid evaporation and sample drying.
6. Wash the cells with 1 ml of 1X Reaction Buffer with Stabilizer Solution.
7. Observe. Depending on the settings, yellow or red-orange punctuate staining will be apparent in healthy cells under the microscope. Diffuse orange fluorescence will appear in the cytoplasm of cells containing damaged mitochondria.
8. Other treatments can also be applied to the cells at this point to evaluate their effects on mitochondrial DYm. Note that mitochondrial reactions occur quickly after insult therefore constant observation or recording may be required to observe the shift.
b. Staining of Suspension Cells
1. Harvest about 1 x 106 cells per sample by centrifugation at 250 x g for 5 minutes at room temperature.
2. Dilute the 10X Reaction Buffer to 1X with distilled water. To increase the stability of the dye for analysis, add 20 ml of Stabilizer Solution for every 1 ml of 1X Reaction Buffer prepared.
3. To dilute the MitoShift™ dye, add 1 μl of dye to 10 μl of 1X Reaction Buffer prepared in step 1 on page 2 (with or without the Stabilizer Solution). Vortex to homogenize. Again, add 1 μl from this dilution to 1000 μl of 1X reaction buffer. A general starting concentration of 100 nM (1:10,000 dilution) is suggested, keeping in mind that optimization may be required according to cell type.
4. Resuspend cells in 1 ml of diluted MitoShift™ solution, prepared in step 3.
5. Incubate samples at room temperature, in the dark for 30-90 minutes to let the dye equilibrate.
6. Centrifuge cells at 250 x g and discard supernatant.
7. Resuspend cells in 1 ml of 1X Reaction Buffer with Stabilizer solution at room temperature.
8. Observe. Depending on the settings, yellow or red-orange punctuate staining will be apparent in healthy cells under the microscope. Diffuse orange fluorescence will appear in the cytoplasm of cells containing damaged mitochondria.
9. Other treatments can also be applied to the cells at this point to evaluate their effects on mitochondrial DYm. Note that mitochondrial reactions occur quickly after insult therefore constant observation or recording may be required to observe the shift

B. Analysis by Flow Cytometry
The use of MitoShift™ in flow cytometry will be appropriate when an endpoint assay can give information about the status of a cell population. Absence of fluorescence in this assay will indicate cells with depolarized mitochondria or dead cells.
1. Harvest about 1 x 106 cells per sample by centrifugation at 250 x g for 5 minutes at room temperature.
2. Dilute the 10X Reaction Buffer to 1X with distilled water.
3. To dilute the MitoShift™ dye, add 1 μl of dye to 10 μl of 1X Reaction Buffer Vortex to homogenize solution. Again, add 1 μl from this dilution to 1,000 μl of 1X reaction buffer and vortex again. A
general starting concentration of 100 nM (1:10,000 dilution) is suggested, keeping in mind that optimization may be required according to cell type.
4. Resuspend cells in 1 ml of diluted MitoShift™ solution, prepared in the previous
step.
5. Incubate samples at room temperature, in the dark for 30-90 minutes to let the dye equilibrate.
5. Wash samples in 1 ml of PBS with centrifugation at 250 x g.
Note: Keep samples shielded from light until analysis. Stained cells can be kept at room temperature until analyzed.
6. Resuspend cells in 1 ml of PBS and analyze promptly by flow cytometry (use a 488 nm argon laser for excitation and FL2 for detection).
8. Optimize setting of apparatus and incubation times as necessary

VI. Controls
A. Healthy Cells
Controls are necessary to evaluate the level of staining in healthy cells. Some cell lines may be too sensitive to pH variations to allow the use of the MitoShift™ assay. Viable counts of a healthy sample stained with trypan blue will also be helpful to assess general viability. Use of Fetal Bovine Serum (FBS) supplemented media to incubate and wash the cells may improve the results for sensitive cells lines or if extended periods of time are necessary to observe results. For flow cytometry and confocal experiments, unstained samples are also necessary to establish the level of autofluorescence of the cells.
B. Disrupted DYm Control
Treatment of cells with certain drugs that disturb the mitochondrial membrane potential provide an appropriate positive control. The valinomycin included in the kit is a potent potassium ionophore and a known apoptosis inducer. Treatment of a cell sample with 100 nM valinomycin diluted in media or reaction buffer, will immediately disrupt the mitochondrial membrane potential and the fluorescence will be releseased from the mitochondria into the cytoplasm. Valinomycin and as an alternative, carbonyl cyanide p-(trifluoro-methoxy) phenylhydrazone (FCCP, 250 nM) are also used to depolarize the mitochondrial potential before addition of mitochondrial activators.